Targeted Metabolomics Resolves Amino Acid and Lipid Specialization Between Pileus and Stipe in Artificially Cultivated Termitomyces upsilocystidiatus

This study presents the first tissue-resolved targeted metabolomic analysis of artificially cultivated Termitomyces upsilocystidiatus fruiting bodies using LC-MS/MS. We identified pronounced metabolic divergence between the pileus and stipe. The pileus was enriched in a nitrogen-recycling and antioxidant module, exemplified by L-citrulline (~13.5-fold higher than stipe, p < 0.01) and urea, while the stipe accumulates sulfur-derived and oxidized metabolites such as L-homocystine (~3.5-fold higher, p < 0.01) and methionine sulfoxide. Lipid profiles further distinguished the two tissues: the pileus featured high levels of linoleic acid-derived oxylipins, including 13(S)-HODE and 12(13)-DiHOME (~9.7-fold and ~303-fold enrichment, respectively, p < 0.01), suggesting a role in signaling and redox buffering. In contrast, the stipe preferentially accumulated oxidized eicosanoids (e.g., 5-oxoETE) and thromboxane B1, indicative of a stress-responsive lipid network. Together, these metabolite-level observations support a tentative “pileus-synthesis/stipe-defense” dual-hub model. This work provides a quantitative metabolic framework for understanding tissue specialization in a symbiotic fungus and offers practical entry points for cultivation optimization and quality control of Termitomyces.